INTERACTION OF TUBULIN AND MICROTUBULE PROTEINS WITH VANADATE OLIGOMERS

Microtubule assembly is known to be regulated by the phosphorylation o f microtubule-associated proteins (MAPs), and is thus sensitive to pho sphatase inhibitors. We have investigated the direct interaction betwe en phosphatase inhibitors (vanadate, sodium fluoride, and okadaic acid ) and microtubule proteins. Vanadate self-assembles into oligomers, pr imarily dimer, tetramer, and decamer in 0.1 M Pipes, pH 6.9. Oligomer concentrations and their direct binding to tubulin and MAPs were deter mined by V-51 NMR. The assembly of microtubule protein (MTP) is strong ly inhibited by decavanadate binding to MAPs and only weakly inhibited by tetravanadate binding to MAPs. Decavanadate will inhibit both MAP2 and tau-induced assembly. Decavanadate binds to MAP2 at 26 sites [K-a greater than or equal to (1.0-1.3) x 10(5) M(-1)]. The mechanism appe ars to involve competitive binding to MAPs, presumably at or near the microtubule binding domains, and reduced affinity for microtubules. Th e assembly of MAP-free, phosphocellulose-purified tubulin (PC-tubulin) is only weakly inhibited by decavanadate, although decavanadate binds to tubulin at four independent sites (K-a greater than or equal to 1. 0 x 10(5) M(-1)). Monomeric vanadate, a strong phosphatase inhibitor, does not interact with tubulin or MAPs, and thus does not bind to the exchangeable nucleotide binding site on tubulin. Sodium fluoride stimu lates both PC-tubulin and MTP assembly by a nonspecific effect, probab ly involving water structure formation. Wyman analysis suggests an abs ence of direct or specific binding to tubulin (d ln K/d ln [NaF] = 0.2 14). NaCl is nearly as effective in promoting assembly of PC-tubulin, but inhibits MTP assembly. Okadaic acid has no effect on the assembly of MTP or PC-tubulin under standard assembly conditions.